Retroviruses, which include many important pathogens of humans and animals, have become important research tools as benign gene delivery vectors, and have potential as therapeutic vectors for gene therapy. Whether pathogenic or therapeutic, the initial infection and subsequent dissemination of the virus depends on efficient entry of the retrovirus into host cells. While a detailed understanding of the mechanisms of viral entry has not been clearly defined for any retrovirus, all retroviruses share a common overall strategy for entry into cells. The initial step of retrovirus entry, the interaction between the viral surface glycoprotein (SU) and a cellular receptor, is complex, involving multiple, noncontiguous determinants in both proteins that specify receptor choice, binding affinity and the ability to trigger conformational changes in the viral glycoproteins. Despite the complexity of this interaction, retroviruses have the ability to evolve the structure of their envelope glycoproteins to use a different cellular protein as a receptor, often a protein that has no obvious homology to the original receptor, and retain efficient entry functions. How do retroviruses do this? Understanding this ability will provide valuable information for antiviral intervention and targeting gene delivery. We hypothesize that: (1) the envelope glycoproteins are organized into functional domains that allow changes to occur in receptor choice by mutation and/or recombination while maintaining a critical level of both receptor binding affinity and the ability to trigger glycoprotein conformational changes to initiate the fusion process; (2) multiple, noncontiguous receptor interaction determinants located in the SU hypervariable domains are required for binding affinity and to restrict or broaden receptor usage; (3) regions outside of the SU hypervariable domains will function to connect receptor binding to triggering the glycoprotein lock mechanism and will be conserved as the virus changes receptor usage. The homologous group of retroviruses, the subgroups A through E (A-E) avian leukosis viruses (ALV), provide a powerful model system to test these hypotheses by supplying highly related viruses that have evolved from a common ancestor to utilize different receptors. Specifically, we aim to: 1. Genetically define functional regions/residues of the subgroup A-E ALV envelope glycoproteins important for receptor binding affinity. 2. Genetically define functional regions/residues of the subgroup A-E ALV glycoproteins important for the specificity of receptor usage. 3. Identify and characterize regions/residues in the ALV glycoproteins important for connecting receptor binding to triggering the conformational changes that initiate the fusion process. 4. Genetically define functional regions/residues of the ALV receptors necessary for binding affinity and triggering a conformational change in the ALV glycoproteins. 5. Test soluble forms of the ALV glycoproteins for the ability to produce crystals suitable for structural studies. PERFORMANCESITE(S) (organization,city, state) Mayo Clinic Rochester Rochester, MN 55905 KEYPERSONNEL. See instructions. Use continuationpages as needed to provide the requiredinformationin the format shown below. Start with Principal Investigator.List all other key personnelin alphabeticalorder, last namefirst. Name Organization Role on Project Mark J. Federspiel, Ph.D. Mayo Clinic Rochester Principal Investigator Pranay D. Khare, Ph.D. Mayo Clinic Rochester Postdoctoral Fellow Deborah C. Melder Mayo Clinic Rochester Senior Technician Disclosure Permission Statement. Applicableto SBIR/STTROnly. See instructions. [] Yes [] No _PHS 398 (Rev. 05/01) Page 2 Form Page2 _ Number pagesconsecutivelyat the bottom throughoutthe application.Do no_u_sttesuffixessuch as 3a, 3b. _ Principal Investigator/Program Director (Last, first, middle): Federspiel, Mark J. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page ......................................................................................................................................................... 1 Description,